This invention relates to methods and compositions for preventing and treating Clostridium difficile disease.
Clostridium difficile, a toxin-producing gram positive bacterium, invades the intestinal tracts of patients whose normal intestinal flora is suppressed due to treatment with broad spectrum antibiotics. The bacterial toxins cause varying degrees of damage to the large intestinal (i.e., colonic) epithelium, and cause a spectrum of illnesses, ranging from mild diarrhea to severe colitis. Because antibiotic treatment induces the onset of C. difficile disease, the associated syndromes are named antibiotic-associated diarrhea and colitis (LaMont, Bacterial Infections of the Colon, Textbook of Gastroenterology, second edition, 1897-1903, 1995).
Three clinical syndromes caused by C. difficile are recognized, based on severity of the infection. The most severe form is pseudomembranous colitis (PMC), which is characterized by profuse diarrhea, abdominal pain, systemic signs of illness, and a distinctive endoscopic appearance of the colon. The case-fatality rate of PMC may be as high as 10%. Antibiotic-associated colitis (AAC) is also characterized by profuse diarrhea, abdominal pain and tenderness, systemic signs (e.g., fever), and leukocytosis. Intestinal injury in AAC is less than in PMC, the characteristic endoscopic appearance of the colon in PMC is absent, and mortality is low. Finally, antibiotic-associated diarrhea (AAD) is the mildest syndrome caused by C. difficile, and is characterized by mild-moderate diarrhea, lacking both large intestinal inflammation (as characterized, e.g., by abdominal pain, tenderness) and systemic signs of infection (e.g., fever). These three distinct syndromes occur in an increasing order of frequency. That is, PMC occurs less frequently than AAC, and AAD is the most frequent clinical presentation of C. difficile disease.
The populations affected by C. difficile are principally hospitalized, elderly patients and nursing home residents who have received broad spectrum antibiotics. Old age, length of hospital stay, underlying illness, and use of antibiotic therapy are recognized risk factors for C. difficile infection (McFarland et al., J. Infect. Dis. 162:678-684, 1990; Bennett, Aging, Immunity, and Infection, 216-229, 1994). A frequent complication of C. difficile infection is relapsing disease, which occurs in up to 20% of all subjects who recover from C. difficile disease. Relapse may be characterized clinically as AAD, AAC, or PMC. There are no specific risk factors or predisposing factors for relapse, but patients who relapse once are more likely to relapse again.
C. difficile produces two exotoxins, Toxin A and Toxin B, which mediate the disease process caused by C. difficile. Toxin A and Toxin B are large (xcx9c300 kDa) extracellular proteins, the active forms of which are believed to be homodimers. The toxins are stably expressed in approximately equivalent amounts from a single chromosomal locus (Mitty et al., The Gastroenterologist 2:61-69, 1994). The toxins have nearly 50% amino acid sequence homology, but are immunologically distinct. The 100 kDa carboxyl-termini of the two toxins contain repetitive oligopeptides, and are involved in carbohydrate receptor binding in vivo. Receptor specificity is believed to mediate tissue and host specificity of toxin action. This region is also more immunogenic than the amino terminus. The amino terminal 200 kDa region contains the enzymatic domain, which is believed to glycosylate the GTP binding proteins Rho, Rac, and Cdc42, thereby preventing their phosphorylation, and leading to a loss of actin polymerization and cytoskeletal integrity (Eichel-Streiber, Trends Micro. 4:375-382, 1996). As a result of the cytoskeletal changes, tight junctions between epithelial cells are lost. The epithelial damage in conjunction with local inflammatory events causes fluid exudation into the gut, manifested as diarrhea (Mitty et al., supra).
By virtue of their inhibition of cytoskeleton structure, both toxins cause the rounding of cells in tissue culture at very low concentrations. The dose that causes morphologic change in 50% of cells (MC50) for Toxin A on IMR90 cells is 0.4 ng/ml and for Toxin B is 3.5 pg/ml (Torres et al., Infect. and Immun. 63:4619-4727, 1995). Toxin A is an enterotoxin that causes fluid accumulation in ligated animal intestinal loops. Although Toxin B does not induce fluid secretion in animal intestinal loops, both it and Toxin A elicit inflammatory changes in vivo and in vitro (Mitty et al., supra). Both toxins are lethal to animals when administered systemically.
The invention provides methods of treating Clostridium difficile disease in human patients. These methods involve percutaneously (e.g., intramuscularly, intravenously, or intraperitoneally) administering to a patient human C. difficile polyclonal immune globulin that neutralizes both Toxin A and Toxin B (hereinafter xe2x80x9cimmune globulinxe2x80x9d) (e.g., 0.01-100 mg/kg body weight). These methods can also include percutaneous administration of a clostridial toxin or toxoid to a patient, to stimulate an anti-C. difficile immune response in the patient. When administered as treatment in affected individuals, the injected immune globulin will also prevent relapse.
Also included in the invention are methods of preventing C. difficile disease in human patients. In these methods, a toxin-neutralizing antibody to a C. difficile toxin or toxoid (e.g., a C. difficile polyclonal immune globulin (e.g., 0.01-100 mg/kg body weight)) is percutaneously (e.g., intramuscularly, intravenously, or intraperitoneally) administered to a human subject at risk of becoming infected with C. difficile. The C. difficile immune globulin used in these methods can be produced, e.g., in a human. These methods can also include percutaneous administration of a clostridial toxin or toxoid containing Toxin A and Toxin B epitopes to the patient.
The invention also provides methods of preventing or treating intestinal clostridial disease in human patients, which involve percutaneously administering a clostridial (e.g., C. difficile) toxin or toxoid to a patient, in the presence or absence of an adjuvant, such as alum. An additional method included in the invention involves administering C. difficile immune globulin, as described above, to rapidly treat or protect a patient, while simultaneously administering toxoid for long-term, active protection by means of stimulation of the patient""s immune system.
All of the prophylactic and therapeutic methods described above can, in conjunction with percutaneous administration, involve mucosal administration, such as oral or rectal administration.
Also included in the invention are methods of producing C. difficile toxoid. These methods involve providing C. difficile bacteria; culturing the bacteria in media containing suitable animal products (e.g., casein products) to generate a culture; co-purifying clostridial Toxin A and clostridial Toxin B from the culture to generate a mixture of co-purified Toxin A and Toxin B; and inactivating the co-purified Toxin A and Toxin B by incubation in formaldehyde at a temperature of about 25xc2x0 C. or less (e.g., 15xc2x0 C. or less, or 5xc2x0 C. or less) to generate the clostridial toxoid. The co-purified Toxin A and Toxin B can be present in the mixture at a ratio in the range of 0.1:1 to 10:1, for example, 2:1. The invention also includes a C. difficile toxoid produced by this method, and a vaccine composition containing this toxoid and 0.012-0.020% formaldehyde. Optionally, this composition can contain an adjuvant, such as alum.
The invention also provides methods of producing human, toxin-neutralizing C. difficile immune globulin. In these methods, C. difficile toxin or toxoid containing, e.g., Toxin A and/or Toxin B, is administered to a human, and C. difficile immune globulin is isolated from the human. C. difficile immune globulin produced using these methods is also included in the invention.
Also included in the invention are methods of identifying a human producing a C. difficile immune globulin. These methods involve obtaining a blood sample from a human vaccinated with a C. difficile toxoid; determining the level of antibodies to C. difficile Toxins A and B in the blood sample by an enzyme-linked immunosorbent assay (ELISA); and determining the level of in vitro cytotoxicity neutralization activity against C. difficile Toxins A and B in the blood sample. Detection of increased levels of antibodies to C. difficile Toxins A and B in the blood sample, and detection of in vitro cytotoxicity neutralization activity against C. difficile Toxins A and B in the blood sample, indicate identification of a human producing a C. difficile immune globulin. In addition to humans that have been vaccinated with a C. difficile toxoid, this method can be carried out with unvaccinated humans to identify good candidates for vaccination.
The term xe2x80x9cC. difficile immune globulin,xe2x80x9d is used herein to describe polyclonal hyperimmune serum raised in subjects (e.g., human volunteers) vaccinated with C. difficile toxoids. The immune globulin contains antibodies that neutralize cytotoxicity and in vivo effects of Toxin A and Toxin B.
The term xe2x80x9cC. difficile toxoidxe2x80x9d is used to describe a C. difficile toxin (Toxin A or Toxin B) or a mixture of C. difficile toxins that have been partially or completely inactivated, for example, by chemical (e.g., formaldehyde) treatment. A toxin is said to be xe2x80x9cinactivatedxe2x80x9d if it has less toxicity (e.g., 100%, 99%, 95%, 90%, 80%, 75%, 60%, 50%, 25%, or 10% less toxicity) than untreated toxin, as measured, for example, by an in vitro cytotoxicity assay or by animal toxicity. Other chemical means for inactivating toxins can be used including, for example, peroxide or glutaraldehyde treatment. Toxoids can also be generated by genetic changes that result in toxin inactivation.
The invention provides several advantages. For example, treatment using the methods of the invention specifically results in inactivation of C. difficile bacterial toxins, without affecting normal intestinal flora. Both C. difficile Toxin A and Toxin B are involved in human disease, and the immunotherapy methods of the invention can be used to target both of these molecules. Recovery using immunotherapy is more rapid than antimicrobial therapy, which targets vegetative bacteria, rather than directing toxin neutralization. The specific neutralization of toxin activity has the advantage of specifically and rapidly inactivating the cause of tissue damage. In addition, a single dose of C. difficile immune globulin, administered percutaneously (e.g., intramuscularly, intravenously, or intraperitoneally), can be used in the methods of the invention, rather than the repeating dosing required for oral administration (Lyerly et al., Infect. and Immun. 59:2215-2218, 1991). The overall dose of C. difficile immune globulin administered percutaneously is lower than the dose required in oral methods, due to the longer half life of injected antibodies, compared to orally administered antibodies (hours vs. weeks or months). Specific antibody therapy also permits continuation of treatment of underlying conditions with antibiotics, which may otherwise have to be withdrawn to permit reconstitution of the intestinal flora and recovery from C. difficile infection. Also, treatment using the methods of the invention prevents the emergence of antibiotic-resistant bacteria. C. difficile disease has been traditionally treated with vancomycin and metronidizole, and use of vancomycin has led to the emergence of vancomycin-resistant enterococcus. Similar problems may be arising from metronidizole treatment. Finally, C. difficile is cultured in the methods of the invention in medium that lacks complex animal products, such as nervous system products, e.g., the animal products in Brain Heart Infusion medium. Media containing such complex animal products have been found to contain the bovine spongiform encephalopathy (BSE) prion. Thus, in not using such medium, the invention provides safety against infection by such agents.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.